The invention relates to a method for the controlled proportioning of liquids by means of a positive-displacement device to dislocate a gas cushion and a reception volume connected thereto which has an aperture to the environment for the reception and discharge of a liquid by dislocating the gas cushion by means of the positive-displacement device.
The proportioning of liquids while dislocating a gas cushion is performed by means of manual or electric pipettes or automatic proportioning devices. The gas cushion is an air cushion, as a rule. The positive-displacement device is mostly designed as a cylinder having a slidable piston therein. The liquid is mostly received into a pipette tip which can be exchanged. Pipette tips are mostly conceived as disposable articles which can be discarded after a single use. They are made of plastic material, as a rule. The suction and expulsion of the liquid by means of the gas cushion has the advantage that this avoids contaminations of the positive-displacement device and the other re-used components of the proportioning device. A gradual contamination by aerosols or any faulty handling of the proportioning device may be counteracted by filters which are preferably integrated in the pipette tip.
The electric air-cushion pipettes and automatic proportioning devices which are known still are very susceptible with regard to the correctness of the liquid volume discharged and the error in proportioning. They do not even detect major deviations from the desired value of the liquid volume to be discharged, which are caused by a leakage in the proportioning system. Errors in proportioning may also arise while liquids are proportioned under a high vapour pressure. Deviations in the temperature, density, viscosity, and surface tension of the liquid to be proportioned may also cause major errors in proportioning which will not be detected. Errors in handling, e.g. air intake because of too early a withdrawal from the liquid during reception or too small a feed volume, also lead to errors in proportioning.
From U.S. Pat. No. 5,895,838, a method has been known for correcting a temperature-dependent dispensing error in dispensing liquids such as in pipetting, and an apparatus for dispensing liquids such as a pipette which has a higher degree of accuracy. According to the method, dispensing is performed by means of two chambers which are connected to each other via a gas passage. The first chamber, in addition to communicating with the gas passage, communicates with the liquid to be dispensed and the second chamber is gas-tight except for the gas passage. To receive a liquid in the dispensing apparatus, the volume of the second chamber is enlarged, which causes an entry of gas into it from the first chamber and, in turn, an inflow of the liquid to be dispensed into the first chamber until a pressure balance is reached between the chambers. A measurement is made of the change of temperature of the gas flowing from the first chamber into the second chamber and the change in volume which is caused in the second chamber is corrected on the basis of the change of temperature as measured so that the desired amount of liquid is received into the first chamber. A temperature sensor is installed in the second chamber in the vicinity of the gas passage to measure the temperature and a sensor which is mounted in the first chamber may be used in addition. This method is only suitable for the correction of temperature-related errors in proportioning.
From DE 35 31 241 A1, a device has been known for the controlled dispensing of liquids which has a cannula to receive the liquid and an axially movable pressure transducer at one end of the cannula where a connection remaining constant specifically in its volume is disposed between the cannula and the pressure transducer. At least one pressure sensor is disposed between the pressure transducer and the cannula, specifically within the connection, and a regulating connection is provided between the pressure sensor and a driving device for the pressure transducer.
Accordingly, it is the object of the invention to provide an improved method for the controlled proportioning of liquids by means of a gas cushion.
The object is attained by a method having the features of claim 1. It further is attained by a method having the features of claim 10. Advantageous aspects of the methods are set forth in the sub-claims.
The point to start from for the invention is that the error in proportioning which is caused by error sources such as deviations of the vapour pressure, temperature, density, viscosity, and surface tension of the liquid to be proportioned, modifications to the geometry of the pipette tip, leakages of the system, and errors in handling will influence the pressure which prevails in the gas cushion while it is dislocated. The invention involves a measurement of the pressure in the gas cushion to comprise all of the aforementioned error sources.
According to the first approach, in a method for the controlled proportioning of liquids by means of a positive-displacement device to dislocate a gas cushion and a reception volume connected to the positive-displacement device which has an aperture to the environment for receiving and discharging a liquid by dislocating the gas cushion by means of the positive-displacement device,
the pressure pab in the gas cushion is measured at the time of a complete discharge of a liquid volume,
the discharge time tab for a complete discharge of the liquid volume is measured, and
the time demand xcex94t to compress the gas cushion by the positive-displacement device is determined by means of the pressure pab, the dead volume Vo of the gas cushion, a gas condition equation, and the volumetric gas flow Q discharged by the positive-displacement device,
a corrected discharge time toab is determined as a proportioning control magnitude by a deduction of the time demand xcex94t from the discharge time tab measured, and
the deviation of the corrected discharge time toab from a desired discharge time tsoll is output and/or is saved and/or is resorted to for regulating the discharge of the liquid volume.
The discharge time tab is that time which the positive-displacement device needs from the beginning to the end of gas cushion dislocation and, hence, to the time at which the liquid volume is completely discharged. The dead volume Vo of the gas cushion is that volume which the gas cushion has at the beginning of liquid reception. The positive-displacement device simultaneously causes the gas cushion to undergo a dislocation and a compression during the discharge time tab. The dis-location of the gas cushion is matched by the liquid volume discharged. The above error sources have an impact on the discharge time and the extent to which the gas cushion is compressed. As a result, the error in proportioning may be characterized by the time demand xcex94t for the compression of the gas cushion. Thus, the corrected discharge time toab which results from the reduction of the discharge time tab by the time demand xcex94t is a control magnitude suited for the proportioning of liquids which allows to discover errors in proportioning. To this effect, the deviation of the corrected discharge time toab from a desired discharge time tsoll may be output, e.g. by signalling the degree of the deviation or, if a tolerable deviation is exceeded, by giving a warning message (e.g. acoustically, optically or in another perceivable form). In addition or instead, the deviation may be saved, e.g. to furnish a proof on how precise a proportioning was. In addition or instead, the deviation may be re-sorted to in order to regulate the discharge of a liquid volume, e.g. by enhancing the accuracy of proportioning from discharge to discharge if the same liquid volume is discharged several times or step by step.
The error in volume (change in volume) xcex94t V may be determined by multiplying the time demand xcex94t by the volumetric gas flow Q.
The beginning of the discharge time tab may be favourably determined with the aid of control signals of a device for controlling a driving motor of the positive-displacement device. The end of the discharge time tab may be favourably determined by measuring a pressure drop in the gas cushion because a significant pressure drop will occur in the gas cushion at the time of complete discharge of the liquid volume. This pressure drop can be measured in both discharging a liquid volume, which was received, in a single discharging step (xe2x80x9cpipettingxe2x80x9d) and stepwise discharging the liquid volume, which was received, in several constant or variable partial steps (xe2x80x9cdispensingxe2x80x9d), after each partial step. This makes possible a simple electronic measurement of the discharge time.
The ideal gas equation may be advantageously utilized as a gas condition equation. When combined with the volumetric flow, the time demand xcex94t to compress the air cushion may then be determined with reference to the condition when under an ambient pressure po, as being:
xcex94t t=(pabxc3x97Vo)/((po+pab)xc3x97Q).xe2x80x83xe2x80x83(1)
The corrected discharge time
toab=tabxe2x88x92(pabxc3x97Vo)/((po+pab)xc3x97Q)xe2x80x83xe2x80x83(2)
has then be corrected to the ambient pressure po. Correction to the ambient pressure po is an advantage as it favours the use of a relative-pressure sensor which measures the pressure differential from the ambient pressure po.
The corrected discharge time toab can be compared to a desired discharge time tsoll which is achieved during proportioning free from proportioning errors or during proportioning including tolerable proportioning errors. In the ideal case where the gas cushion does not undergo compression the volume dislocated by the positive-displacement device is accurately equal to the liquid volume discharged. Accordingly, the desired discharge time tsoll can be calculated as a quotient of the desired volume Vsoll of the liquid to be discharged and the volumetric gas flow Q delivered by the positive-displacement device.
The correctness and precision of a proportioning device may be verified by weighing liquid volumes which were discharged. The results of these measurements may be utilized for a calibration of the proportioning device. For example, this can be accomplished in such a form that the desired discharge time is calculated as a product of the quotient from the desired volume Vsoll and the volumetric gas flow Q using a calibration constant Ccal where the calibration constant Ccal is determined because of a calibration of the proportioning device. Then, the desired discharge time tsoll will be calculated as being:
tsoll=Ccalxc3x97Vsoll/Q.xe2x80x83xe2x80x83(3)
The volumetric gas flow Q delivered by the positive-displacement device can be determined by means of the effective surface Fkol, and the speed Vkol of a positive-displacement element of the positive-displacement device, e.g. a piston of the piston-and-cylinder unit:
Q=Fkolxc3x97vkol.xe2x80x83xe2x80x83(4)
An advantageous aspect of the method takes into account that the displacing speed of the positive-displacement device is not constant during the discharge time, but may be subjected to acceleration up to a constant final speed. Another advantageous aspect of the method takes into account that the positive-displacement device may have a radial clearance which needs balancing upon reversal of the direction of delivery of the positive-displacement device. This is taken account of by the method by the fact that either an additional time demand tbe for an acceleration to a constant final speed and/or an additional time demand ts for a radial clearance of the positive-displacement device that requires balancing during the positive displacement is deducted from the corrected discharge time toab or such an additional time demand tbe and/or ts is added to the desired discharge time tsoll.
The corrected discharge time toab may be determined in a real time during the pipetting operation. A comparison of the corrected discharge time toab to the desired discharge time tsoll allows a very good control of the accuracy in proportioning. It is particularly the desired volume Vsoll of the liquid and the speed of displacement vkol of the positive-displacement device which enter the desired discharge time tsoll as variables. All of the other variables such as the geometry of the pipette tip, the viscosity of the liquid, etc. are eliminated in this comparison.
According to the second approach, in a method for the controlled proportioning of liquids by means of a positive-displacement device to dislocate a gas cushion and a reception volume connected to the positive-displacement device which has an aperture to the environment for receiving and discharging a liquid by dislocating the gas cushion by means of the positive-displacement device,
the pressure p in the gas cushion is measured during a dislocation of the gas cushion,
the volume variation xcex94t V of the gas cushion is determined by means of a gas condition from the pressure p as measured in the gas cushion,
the corrected liquid volume Vflxc3xcss discharged is calculated by a deduction of the volume variation xcex94t V of the gas cushion from the gas volume Vgas dislocated by the positive-displacement device at the time t of measuring the pressure p, and
the positive-displacement device is stopped when the corrected liquid volume Vflxc3xcss discharged corresponds to a desired volume Vsoll, or
the corrected liquid volume Vflxc3xcss discharged is calculated at the pressure pab and the time tab at the time of a complete discharge of a liquid volume and is compared to a desired volume Vsoll and the deviation of the corrected liquid volume Vflxc3xcss from the desired volume Vsoll is output and/or is saved and/or is resorted to for regulating the discharge of the liquid volume.
In this method, the volume variation xcex94t V of the gas cushion is calculated by means of the pressure p measured and the gas volume Vgas dislocated by the positive-displacement device is determined and the corrected liquid volume Vflxc3xcss discharged is calculated by deducting the volume variation xcex94t V from the gas volume Vgas dislocated. This can be accomplished either permanently or repeatedly through-out the discharge of the liquid and can release the discontinuance of the discharging operation once the corrected liquid volume Vflxc3xcss discharged corresponds to a desired volume Vsoll. However, it is also possible to determine the corrected liquid volume Vflxc3xcss discharged for the pressure pab at the time of a complete discharge of the liquid volume and the time tab measured therefor and to compare the volume to a desired volume Vsoll. The deviation of the corrected liquid volume Vflxc3xcss discharged from the desired volume Vsoll, in turn, can be output and/or can be saved and/or can be resorted to for regulating the discharge of the liquid volume as is described above for the deviation of the corrected discharge time toab from the desired discharge time tsoll. 
According to another advantageous aspect of the method, the volume variation xcex94t V of the gas cushion is determined as being
xcex94t V=pxc3x97Vo/(po+p)xe2x80x83xe2x80x83(5)
by means of the value measured for the pressure p in the gas cushion and the ideal gas equation. The gas volume Vgas dislocated by the positive-displacement device at the time t of measuring the pressure p can be determined in various ways. Thus, for example, this can be accomplished by measuring the path s which is travelled by a positive-displacement element of the positive-displacement device from the beginning of displacement up to the time t, and by multiplying this path s by the effective surface fkol of the positive-displacement device which, in particular, may be a piston surface. In a positive-displacement device which has a stepping motor, the xe2x80x9cpath measurementxe2x80x9d may also be accomplished by counting the number n of steps with the path s resulting from a multiplication of the steps n by the step size xcex94t s. In addition, the gas volume Vgas dislocated may be determined by a measurement of the time t from the beginning of gas cushion dislocation up to the measurement of the pressure p. As in the measurement described above, the beginning of this time t may be advantageously released by control signals of a device for controlling a driving motor of the positive-displacement device. If a sufficiently accurate assumption is made that the positive-displacement device supplies a constant volumetric gas flow Q the corrected liquid volume Vflxc3xcss discharged can be calculated as being:
xe2x80x83Vflxc3xcss=txc3x97Q xe2x88x92xcex94V.xe2x80x83xe2x80x83(6)
According to what was set forth above the volumetric gas flow Q delivered may be determined by means of the effective surface F and the speed Vkol of a positive-displacement element of the positive-displacement device. In case of need, an additional time demand tbe for an acceleration to a constant final speed and/or an additional time demand ts for a clearance of the positive-displacement device that requires balancing during the positive displacement may be deducted from the time t.
In an aspect of this regulated proportioning, the positive-displacement device is stopped in such a way that the liquid volume Vflxc3xcss plus a residual amount of liquid Vrest flowing on after the stoppage corresponds to the desired volume Vsoll of the liquid to be discharged:
Vsoll=Vflxc3xcss+Vrestxe2x80x83xe2x80x83(7)
Empiric values may be resorted to for the residual amount Vrest.
The methods described previously relate to the discharge of liquids which already are in the volume to be received. According to an aspect of the method, a liquid volume is received before which corresponds as exactly as possible to the desired volume to be discharged which requires to be discharged by one discharge step or corresponds as exactly as possible to the desired volumes which require to be dispensed by several steps. For example, this may be ensured by designing the proportioning device according to one of the methods which are referred to in EP 0 562 358 B 1 as belonging to the state of the art or as forming the subject matter of this patent. This may further be accomplished by means of measuring the pressure in the gas cushion while the liquid is received. At this stage, the increase of the volume of the gas cushion with regard to the dead volume Vo due to the weight of the liquid column drawn in is calculated by means of the gas condition equation with reference to the pressure measured and the liquid volume really received is calculated taking into account the geometry of the reception volume. The reception of liquid may be regulated until the desired liquid volume is received.
As far as the above methods provide for a regulation of the discharge of liquid volume it is possible to dispense with a liquid reception which is as exact as possible. Preferably, it has to be ensured here that at least the desired volume to be discharged or the sum of the desired volumes to be discharged should be received.
The methods described above are meant for use in proportioning devices having motor-driven positive-displacement devices, particularly for use in electric pipettes or automatic proportioning devices.